CD34+ cells modified with mesenchymal cell mitochondria

Abstract

The invention relates to CD34+ hematopoietic stem cells modified via enrichment with mitochondria isolated from mesenchymal stem cells (MSCs) in an oxidative phosphorylation (OXPHOS) metabolic state. Mesenchymal cells are preferably obtained from the umbilical cord. The CD34+ cells modified in this way exhibit improved functional properties, including increased viability, resistance to oxidative stress, and enhanced hematopoietic transplantation capacity. The invention also relates to a method for mitochondrial transfer to CD34+ cells, as well as pharmaceutical compositions comprising said cells for use in hematological and autoimmune therapies, specifically in bone marrow transplants.

Description

[0001] CD34+ CELLS MODIFIED WITH MITOCHONDRIA FROM MESENCHYMAL CELLS

[0002] Descriptive report

[0003] Scope

[0004] The present invention falls within the field of medical biotechnology and cell engineering, and more specifically within the field of advanced cell therapies and hematopoietic stem cell transplantation. The invention relates to CD34+ hematopoietic stem cells + modified by incorporating or transferring mitochondria from mesenchymal stem cells (MSCs), with the aim of improving their viability, regenerative capacity, tolerance to oxidative stress and / or efficiency in hematopoietic reconstitution after their administration in clinical procedures.

[0005] Background of the invention.

[0006] Hematopoietic stem cells (HSCs) constitute a subpopulation of multipotent progenitor cells capable of self-renewal and differentiation into all hematopoietic cell lineages. Within this group, CD34 cells + They represent a widely accepted phenotypic marker for the identification and isolation of these cells, since it is a transmembrane glycoprotein expressed in immature hematopoietic stem and progenitor cells.

[0007] CD34 cells + They can be obtained from various biological sources, including bone marrow, peripheral blood, and umbilical cord blood. These cells are essential in hematopoietic transplantation procedures, both autologous and allogeneic, as they have the capacity to fully and functionally reconstitute the recipient's hematopoietic system after its destruction in therapies such as chemotherapy or radiotherapy.

[0008] Mitochondria in CD34 cells +

[0009] The role of mitochondria (MT) in the fate and function of CD34 hematopoietic cells +It has been described as a regulator of self-renewal, differentiation, and engraftment. Blood is considered a highly regenerative tissue composed of short-lived cells. Hematopoietic cells must be constantly replenished, with approximately one billion cells being produced daily. Due to this highly dynamic process, blood requires stem cells that can self-renew and simultaneously mobilize and differentiate into mature cells. Hematopoietic stem cells (HSCs) can meet these requirements using metabolic plasticity. Under homeostatic conditions, adult HSCs are inactive cells that rely primarily on anaerobic glycolysis for energy production. On the other hand, differentiated progenitor cells (HPCs) have a rapidly increasing energy demand that must be met.For this reason, differentiation is accompanied by a change to aerobic respiration, or mitochondrial respiration, and an increase in mitochondrial activity.

[0010] The molecular regulation of metabolic and bioenergetic pathways is fundamental in coordinating a cooperative response and other regulatory mechanisms for the maintenance of human stem cells (HSCs) and lineage differentiation. All evidence suggests that even when HSCs depend on glycolytic metabolism, the integrity of their mitochondria, mitochondrial compartments, and metabolites is vital for their fate and function.

[0011] Based on this background, the inventors have proposed that transplanting mitochondria derived from metabolically different cells would allow the regulation of the fate and function of HSPCs (multipotent hematopoietic progenitor cells) in vitro and in vivo, offering an alternative therapy in cases of leukemia, myeloma, lymphoma, or other autoimmune or hereditary blood diseases where CD34 cell grafts are performed. + (HSPCs) for bone marrow repopulation. To test the hypothesis, studies were performed in a non-irradiated immunodeficient mouse model with competitive repopulation.

[0012] One of the problems with HSPC treatments is the limited number of cells that can be obtained. Current therapies require 2 million CD34 cells. +per kilogram of patient to be treated, to ensure adequate hematological recovery post-transplant, which requires a large expansion of the obtained cells. Efficient expansion, without the cells differentiating (i.e., maintaining their primitive phenotype), has been one of the limiting factors in these treatments, and is one of the advantages offered by the modified cells and their method of obtaining and applying them, according to the invention.

[0013] In the current state of the art, mitochondrial transplantation into various cell types is known, and mitochondrial transplantation into HSPCs has even been described, for example, in the work of Jacoby, Elad, et al. "Mitochondrial augmentation of CD34+ cells from healthy donors and patients with mitochondrial DNA disorders confers functional benefit." NPJ Regenerative Medicine 6.1 (2021): 58, where mitochondrial transplantation from healthy donors is performed as a treatment for patients with mitochondrial disorders. Therefore, this document does not anticipate mitochondrial transplantation into healthy cells, specifically HSPCs, to improve their performance in a HSPC graft in a patient with leukemia, myeloma, lymphoma, or other autoimmune or hereditary blood disorders requiring bone marrow repopulation.

[0014] DESCRIPTION OF THE FIGURES

[0015] Figure 1. Artificial mitochondrial transfer of CD34+ cells depending on the dose and hematopoietic subpopulation. (A) Schematic illustration of the contact-independent artificial mitochondrial transfer protocol. (B) Representative confocal laser scanning microscopy images of UCB-CD34 cells + Human Mitotracker+ cells after 24 hours of MitoCeption with isolates containing UC-MSC-derived mitochondrials; scale bar, 2 pm. The mitochondrial dose used was 1.0 million UC-MSCs per million cells (1:1 ratio). (C) Frequency and (D) Mean fluorescence intensity (MFI) of the MitoTracker Green+ healthy donor HSC subpopulation (live / CD34 + / CD38 ) after 24 hours of MitoCeption with UC-MSC-derived mitochondrial-containing isolates in different donor cell ratios (1:10, 1:5, 1:3 and 1:1).

[0016] (E) Frequency and (F) Mean fluorescence intensity (MFI) of the HPC subpopulation of healthy MitoTracker Green+ donors (live / CD34 + / CD38 + (G) Frequency of preclassified HSCs from healthy donors MitoTracker Deep Red+ (live / CD34) + / CD38 ) and HPC (live / CD34 + / CD38 + ) stained with CD34 +(Green) after 24 hours of MitoCeption with MitoTracker Deep Red derived from UC-MSC mitochondria (red) and analyzed by CSLM; 75 focal planes were analyzed for HSCs and 25 focal planes for HPCs. A total of 3 different HSPC and UC-MSC donors (p. 4-6) were used in three independent experiments. All data are presented as mean ± SEM. Statistical significance was assessed using the unpaired Kruskal-Walli nonparametric test with Dunn's multiple comparison test (3 experimental groups). *P < 0.05, **P < 0.005, ***P < 0.0001.

[0017] Figure 2. OXPHOS-derived mitochondrial donation modulates mitochondrial content and membrane potential in CD34 subpopulations +(A) Schematic illustration of a metabolically different mitochondrial transfer protocol. (B) Representative transmission electron microscopy images of HSPCs that did not receive mitochondria (HSPCS alone), that received mitochondria derived from untreated UC-MSCs (HSPC + MITOoxphos), or from UC-MSCs treated with OLN-A (HSPC + MITOglico). Arrows show mitochondria; scale bar, 1 pm. The mitochondrial dose used was 1.0 million UC-MSCs per million cells over 24 hours. (C) Quantification of the number of mitochondria per cell per experiment and mitochondrial area per cell per experiment.

[0018] (E) Low and high TMRE frequency in the LT-HSC subpopulation (CD34 + CD38 CD133 + ) after 24 hours of incubation with pellets containing MITOoxphos or MITOglico. (F) Representative FACS graphs depicting A^Pm (black boxes) of LT-HSC (CD34 + CD38 CD133 +(G) alone (left), MITOoxphos (center) or MITOglico (right). (G) Mean fluorescence intensity (MFI) of low and high TMRE in the LT-HSC subpopulation (CD34 + CD38 CD133 + ) after 24 hours of incubation with pellets containing MITOoxphos or MITOglico (H) Low and high TMRE frequency in the ST-HSC subpopulation (CD34 + CD38 CD133) after 24 hours of incubation with pellets containing MITOoxphos or MITOglico. (I) Representative FACS graphs depicting A^Pm (black boxes) of ST-HSC (CD34 + CD38 CD133) alone (left), MITOoxphos (center) or MITOglico (right). (J) Mean fluorescence intensity (MFI) of low and high TMRE in the ST-HSC subpopulation (CD34 +CD38 CD133) after 24 hours of incubation with pellets containing MITOoxphos or MITOglico. Three different donors of multipotent hematopoietic progenitor cells (HSPCs) and umbilical cord mesenchymal stem cells (UC-MSCs) were used in three independent experiments. All data are presented as mean ± standard error of the mean (SEM). Statistical significance was assessed using the non-parametric Kruskal-Walli test and Dunn's multiple comparisons test. *P < 0.05, **P < 0.005, ***P < 0.0001.

[0019] Figure 3. OXPHOS-derived mitochondrial donation promotes CD34 cell expansion + (HPC).

[0020] (A and B) Day 1 to Day 7 Early Cell UCB-e-CD34 Augmentation Factor + (hu) and with long-term reconstitution activity UCB-LT-CD34 + (hu) (CD34 + CD38 + / CD34 + CD133 + ). UCB-CD34 cells +(hu) were cultured with mitochondria derived from MSC-UC cells: MITOoxphos and MITOglico. After 24 hours, the cells were collected, counted, and seeded (3000 per well). The UCB-CD34 cells + (hu) cells were cultured in StemSpan™ complete medium supplemented with a cytokine mixture containing SCF, TPO, FLT3, and IL-6 for 7 days; the medium was replenished daily. Three mitochondrial donors from MSC-UC (hu) cells and three donors from UCB-C34 cells were used. +(hu) in three independent experiments. The mitochondrial dose used was 1.0 million (u) of UC-MSCs per million recipient cells for 24 hours. Data in (B) are presented as mean ± SEM. Statistical significance was assessed using a two-way RM ANOVA with Geisser-Greenhouse correction and Dunnett's multiple comparisons test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. (C) Cell fraction from each sample for two groups (HSCs and MMPs) after single-cell transcriptomic sequencing and cell pooling. All samples were normalized to the same number of cells. UCB-CD34+ cells were cultured with MITOoxphos and MITOglico. After 24 hours, cells were harvested and assessed by single-cell lOx sequencing.

[0021] Figure 4. OXPHOS-derived mitochondrial donation in CD34 cells +Promotes the differentiation of hematopoietic stem cells in vitro. (A) Total number of colonies observed after 10 to 12 days of culture in semisolid media. The total number of HSPC colonies is shown after donation of UC-MSC-derived mitochondria without MITOoxphos treatment or treated with OLN-A for 24 hours, MITOglico. (B) Representative light microscopy images of type-specific colonies after donation of Mitooxphos or MITOglico. (CE) Percentage distribution of colonies according to type: BFU-E (Blast-forming unit-erythrocyte), CFU-M (Colony-forming unit-monocyte), CFU-G (Colony-forming unit-granulocyte), CFU-GM (Colony-forming unit-granulocyte, monocyte) and CFU-GEMM (Colony-forming unit-granulocyte, erythrocyte, monocyte, megakaryocyte) for HSPCs (C) only, (D) MITOoxphos (center), or (E) MITOglico (right); Scale bar, 100 pm.Three different HSPC and UC-MSC donors were used in three independent experiments. Data in A are presented as mean ± SEM. Statistical significance was assessed using the non-parametric Kruskal-Walli test and Dunn's multiple comparisons test. *P < 0.05; **P < 0.005; ***P < 0.0001.

[0022] Figure 5. OXPHOS-derived mitochondrial donation in CD34 cells +Promotes ROS clearance in HSCs in vitro. (A, B) Mean fluorescence intensity (MFI) of the total ROS dye CM-H2DCFDA in (A) HSCs and (B) HPCs, after 24 hours of incubation with untreated mitochondrial pellets MITOoxphos or OLN-A-UC-MSC derivatives, MITOglico. The mitochondrial dose used was 1.0 million UC-MSCs per million cells for 24 hours. Three different donors of HSPCs and UC-MSCs were used in three independent experiments. All data are presented as mean ± SEM. Statistical significance was assessed using the non-parametric Kruskal-Walli test and Dunn's multiple comparisons test. *P < 0.05; **P < 0.005; ***P < 0.0001.

[0023] Figure 6. Donation of OXPHOS-derived mitochondria improves CD45 cell engraftment + total in peripheral blood. (A) Schematic illustration of the in vivo huCD34 cell graft-and-reconstitution assay +total in NOD-SCID mice. (B) Representative FACS plots of total CD45 cell grafting + (C) FACS analysis of the human total hematopoietic stem cell graft in mouse cardiac blood 6 weeks after transplantation. (D) Representative FACS plots of the total CD45 stem cell graft +Human hematopoietic stem cells in mouse cardiac blood 8 weeks post-transplant. (E) FACS analysis of the graft of total human hematopoietic stem cells in mouse cardiac blood 8 weeks post-transplant. Forty-two mice and three different umbilical cord blood units were used in three independent experiments (sham - 6; HSPCs alone - 9; HSPCs + MITOoxphos - 15; HSPCs + MITOglico - 12). All data are presented as mean ± SEM. Statistical significance was assessed using the non-parametric Kruskal-Walli test and Dunn's multiple comparisons test. *P < 0.05; **P < 0.005; ***P < 0.0001.

[0024] Figure 7. OXPHOS-derived mitochondrial donation enhances CD45 hematopoietic stem cell engraftment + total, but not of lineage-negative hematopoietic stem cells in the spleen. (A) Representative FACS charts of total human hematopoietic cells (CD45 +) in the spleen. (B) Frequency (%) of total CD45 cell engraftment + Human cells were transplanted into mouse spleens after transplantation. The mitochondrial dose used was 1.0 million UC-MSCs per million cells for 24 hours. The cellular dose used was 65,000 HSPCs (CD34+). + ) per individual. Forty-two mice and three different umbilical cord blood units were used in three independent experiments (sham - 6; HSPCs alone - 9; HSPCs + MITOoxphos - 15; HSPCs + MITOglico - 12). All data are presented as mean ± SEM. Statistical significance was assessed using the non-parametric Kruskal-Walli test and Dunn's multiple comparisons test.*P < 0.05; **P < 0.005; ***P < 0.0001.

[0025] Figure 8. OXPHOS-derived mitochondrial donation improves CD45 cell engraftment + total in bone marrow. (A) Representative FACS charts of total human hematopoietic cells (CD45 +(B) Frequency (%) of cell engraftment (CD45) + ) total human in mouse bone marrow after transplantation. DESCRIPTION OF THE INVENTION

[0026] The invention relates to a method for improving the properties of CD34 cells + (HSPC Multipotent hematopoietic progenitor cells), by transplanting them with mitochondria (Mito; MT) from mesenchymal stem cells (MSC) in a state of oxidative phosphorylation (OXPHOS; oxp).

[0027] The cells obtained by the method of the invention, CD34 cells + Mito-OXPHOS cells have better properties when used in bone marrow grafting: they result in faster engraftment and a higher percentage of total hematopoietic lineage than that obtained with CD34 cells. +wild, or unmodified. Thus, these cells obtained by the treatment of the invention are useful in grafts for the reconstitution of bone marrow after its ablation in the treatment of diseases such as: leukemia, myeloma, lymphoma or other autoimmune or hereditary blood diseases.

[0028] HSPC cells are identified by the CD34 marker + , and include the following subpopulations:

[0029] - Hematopoietic stem cells (HSCs): They are multipotent and have the ability to self-renew and differentiate into all blood cells.

[0030] - Hematopoietic progenitor cells (HPCs): They have a lower capacity for self-renewal and are more committed to specific lineages.

[0031] As indicated in the background of the invention, these cells are used in the treatment of diseases such as leukemia, myeloma, lymphoma, and other blood disorders, where it is important that they engraft in the bone marrow and act as healthy hematopoietic cells, restoring the function lost due to the pathology. To improve their functional restoration capabilities, it is essential to enhance their engraftment capacity and ability to remain functional, as well as to achieve better proliferation and differentiation promotion of CD34 cells. + in early progenitors. To promote these attributes, the inventors proposed enriching CD34 cells +Healthy cells with functional mitochondria obtained from mesenchymal stem cells. Mesenchymal stem cells (MSCs) are a key tool in cell therapies due to their multipotency, immunomodulatory properties, and ease of obtaining them. These cells can be isolated from various tissue sources. Among the most commonly used are MSCs derived from bone marrow and those obtained from the umbilical cord, which exhibit high proliferation and low immunogenicity, making them especially useful in regenerative medicine and immunotherapy. MSCs from amniotic fluid, dental pulp, amniotic membrane, adipose tissue, or other MSC sources can also be used.

[0032] Although the inventors used only one type of mesenchymal stem cell in the examples, there is no reason to think that mitochondria from other mesenchymal cells would behave differently from that obtained with umbilical cord stem cells.

[0033] In the experiments, two options were used to transfer mitochondria between mesenchymal cells and CD34 cells. + The first option is to co-culture either the mesenchymal cells from step a) or the mitochondria-rich pellet from step a1a); both cell types are where mitochondrial transfer occurs spontaneously. The second option, to isolate the multiple effects of cell-cell contact or paracrine effect, which would occur in a direct mitochondrial transfer (MitoT) between donor MSC cells and CD34 cells + For recipients, artificial Mitoception or MitoT is used, where mitochondria are isolated from donor UC MSC cells and the pellets containing them are what make contact with the recipient cells or CD34 cells + .

[0034] Tests were performed on mitochondria obtained from stem cells (MSCs) in two metabolic states: glycolytic metabolism and oxidative phosphorylation (OXPHOS) metabolism. To induce glycolytic metabolism, the MSCs were pretreated with oligomycin.

[0035] For an expert in the technique, it will be evident that when discussing the metabolic state of a cell population, this corresponds to the majority state of that cell population, since there is always a degree of coexistence of different metabolic pathways, even within the same cell. Therefore, when we speak of glycolytic metabolism or oxidative phosphorylation, these are the predominant states of the cell population.

[0036] Glycolytic metabolism is a metabolic process in which cells generate energy primarily through glycolysis. This process does not require oxygen, is anaerobic, and produces a limited amount of energy in the form of ATP. This type of metabolism is common in CD34+ hematopoietic stem cells (HSCs). + quiescent cells need to maintain their self-renewal potential and avoid oxidative stress. This is because, by not depending on mitochondrial respiration, glycolytic metabolism generates fewer reactive oxygen species (ROS), thus reducing oxidative damage.

[0037] As will be seen in the examples, this approach, focused on the most suitable metabolism for CD34 cells + HSPCs, did not have good results.

[0038] Surprisingly, mitochondria obtained in the basal metabolic state or OXPHOS of MSCs showed positive effects on CD34 cells +(HSC hematopoietic stem cells) and HPC (hematopoietic progenitors), obtaining significant differences between both groups of mitochondria.

[0039] Contrary to expectations, mitochondria from MSC cells induced to glycolytic metabolism were observed to induce an increase in ATP production in CD34 cells + and did not promote the expansion or efficient differentiation of CD34 cells + .

[0040] In contrast, mitochondria from MSC cells in OXPHOS metabolism did promote a glycolytic metabolic profile in recipient HSCs, favoring their expansion and differentiation into early progenitors, decreasing oxidative stress.

[0041] Thus, it is observed that the glycolytic metabolism of CD34 cells +It is best induced with mitochondria from cells that are not in the same type of metabolism being sought. The results after mitochondrial transfer show that, indeed, CD34 cells + Those that received OXPHOS mitochondria showed a higher number of mitochondria per cell (average of 21 mitochondria) compared to untreated HSCs (average of 8 mitochondria).

[0042] CD34 cells + Cells that received OXPHOS mitochondria showed a lower NADH fluorescence half-life (r - 0.59), indicating that a preferentially glycolytic metabolism was induced, which is favorable for CD34 cells + quiescent. Likewise, CD34 cells + HSCs treated with OXPHOS mitochondria maintained a low mitochondrial polarization (A^Pm), characteristic of quiescent HSCs with high grafting capacity.

[0043] One of the main disadvantages of using CD34 cells+ The problem is that a limited number of these stem cells are obtained from umbilical cord blood, which has restricted their use primarily to pediatric transplants. Optimized culture conditions using serum-free media with a cytokine mix of thrombopoietin (TPO), stem cell factor (SCF), flt3 ligand (TPO), and interleukin-6 (IL-6) promote robust HSC proliferation but also trigger differentiation, leading to reduced HSC activity. This differentiation often involves the loss of the surface proteins CD34 and CD133, markers of HSCs and progenitor cells. Therefore, a method that promotes expansion without inducing differentiation is desirable. The results show (see Example 6) that CD34 cells effectively + MSC-modified cells with MITOoxphos promote expansion without inducing differentiation, while maintaining the CD34 marker + .

[0044] The most important attribute sought in this invention is to improve the engraftment capacity of these cells, and indeed, the CD34 cells + Cells treated with OXPHOS mitochondria showed a significantly greater capacity for bone marrow engraftment compared to CD34 cells + untreated or treated with mitochondria obtained from oligomycin A-treated cells (OLN-A), where mitochondria from glycolytic metabolism (MITOglico) are obtained. CD34 cells + Cells treated with OXPHOS mitochondria also showed an increase in the production of mature cells, with a greater production of CD3 lymphocytes + , CD4 + and CD19 + in peripheral blood.

[0045] It was observed that mitochondrial transfer derived from donor cells in OXPHOS metabolism (MITOoxphos) promoted glycolytic metabolism and a lower A^m compared to the group that received MITOglico, mitochondria derived from oligomycin-treated donor cells (OLN). CD34 cells + that received OXPHOS-derived mitochondria have an enhanced proliferation capacity and promote the differentiation of HSPCs into early progenitors.

[0046] OXPHOS-derived mitochondria helped alleviate ROS following oxidative stress induction, indicating that MitoT is a powerful effector of various vital processes for CD34 cells + Finally, the engraftment and repopulation capacity of CD34 cells was examined. + In vivo after MitoT in a non-irradiated immunodeficient mouse model with competitive repopulation, CD34 cells were shown to be present. +HSPC, which received MT derived from OXPHOS led to faster and greater engraftment of total hematopoietic cells in the recipient mouse in medullary and extramedullary organs.

[0047] CONCLUSION

[0048] Supplementation with healthy mitochondria, mitochondrial components, or mitochondrial-targeted drugs to enhance cellular energy and functional parameters has shown great promise. This invention discloses and protects the use of mitochondrial transfer from MSCs to modulate the fate and function of CD34+ cells. + HSPC. This invention provides evidence that OXPHOS-derived mitochondrial transfer can induce differentiation, proliferation, and ROS clearance of CD34 cells +Finally, it is demonstrated that OXPHOS-derived mitochondrial transfer enables improved engraftment of CD45* cells in a NOD-SCID model. These results are important in the context of HSCT (Hematopoietic Stem Cell Transplantation), where the main challenges are described as delayed engraftment and poor immune reconstitution. The invention provides CD34 cells + with improved properties for grafting in a patient with hematopoietic pathology, where grafting is favored and improved activity is seen in the production of progenitor cells in the recipient organism.

[0049] Thus the invention relates firstly to a CD34 hematopoietic stem cell + modified, where the cell is enriched with mitochondria isolated from mesenchymal stem cells, preferably umbilical cord MSCs, which are in oxidative phosphorylation metabolism. This CD34 cell +The modified cell has improved properties compared to the unmodified cell; for example, it expands in cell culture without altering its HSPC progenitor cell phenotype; it has improved short- and long-term reconstitution and engraftment capacity; and it possesses greater tolerance to oxidative stress (ROS) compared to CD34 cells. + unmodified; and has a higher proportion of bone marrow graft.

[0050] In a second aspect, the invention relates to a method for improving the properties of CD34 cells + , which comprises the following steps: a) obtaining a mesenchymal cell culture in early passages, in a metabolic state of oxidative phosphorylation; a) optionally extracting the mitochondria from the MSC culture, obtaining a pellet rich in mitochondria of MSCs in oxidative phosphorylation (MITOoxphos); b) in parallel obtaining a CD34 cell culture +where these cells do not exhibit deficiencies in their own mitochondria; c) incubate CD34 cells either with the mesenchymal cells from step a) or with the mitochondria-rich pellet from step a1a); and d) obtain CD34 cells + Enriched in mitochondria with enhanced properties of the invention; wherein all stages are carried out under human cell culture conditions for use in cell therapy. CD34 cells + Mitochondria-enriched cells have at least 50% more mitochondrial content compared to untreated cells, and conveniently 100% more mitochondrial content. CD34 cells + They can be of any origin, especially peripheral blood or umbilical cord blood. Likewise, mesenchymal cells can be from umbilical cord blood or any other origin.

[0051] Conveniently in stage c) the ratio between CD34 cells +and the mitochondria ratio is between 1:1 to 1:5 mitochondrial donor cells, in relation to CD34 cells + ; and the incubation of CD34 cells + and MITOoxphos develops for between 12 and 36 hours, especially for 24 hours.

[0052] In a third aspect, the invention relates to a pharmaceutical composition comprising a suspension of CD34 cells + Enriched with mitochondria isolated from mesenchymal stem cells undergoing oxidative phosphorylation metabolism, according to the invention, in a pharmaceutically acceptable vehicle. This composition is used in the treatment of hematopoietic diseases, specifically leukemia, myeloma, lymphoma, or other autoimmune or hereditary blood disorders, and is used in bone marrow transplantation.

[0053] Additionally, any pharmaceutically acceptable carrier is any isotonic infusion solution compatible with live cells, which maintains the viability, functionality and sterility of the cells during storage, transport and administration, for example, but not limited to isotonic saline solution (0.9% NaCl), patient plasma (autologous) or human plasma, which may be supplemented with 1-5% human albumin, heparin, buffer such as PBS, etc.

[0054] The culture conditions for human cells intended for cell therapy must comply with Good Manufacturing Practices (GMP) standards to ensure the quality, safety, and traceability of the cell product. Culture is performed in controlled environments, such as ISO 5 to ISO 7 cleanrooms, with sterile procedures carried out in Class II laminar flow hoods. Cells are cultured at 37 °C in a humidified atmosphere with 5% CO2 and 5% O2. Culture media free of animal-derived components (xeno-free) or human-derived components are used, supplemented with recombinant growth factors, human albumin, or autologous serum.

[0055] Among the blood diseases in which a bone marrow transplant may be useful after bone marrow ablation treatment, we find:

[0056] Malignant hematological diseases: leukemia, myeloma, lymphoma;

[0057] Non-malignant hematological diseases: Severe aplastic anemia; Fanconi anemia; Dyskeratosis congenita; Shwachman-Diamond syndrome; Blackfan-Diamond anemia; Congenital amegakaryocytic thrombocytopenic purpura; Severe congenital thrombocytopenia;

[0058] Hemoglobinopathies and hemoglobin disorders: Sickle cell anemia (drepanocytosis); Thalassemias major (mainly thalassemia major); Pyruvate kinase deficiency;

[0059] Congenital errors of metabolism and lysosomal diseases: Hurler disease (MPS I); Maroteaux-Lamy disease (MPS VI); Metachromatic leukodystrophy; X-linked adrenoleukodystrophy (cerebral form); Krabbe disease; GM1 gangliosidosis; Niemann-Pick disease type B (in some cases)

[0060] Severe primary immunodeficiencies: Severe combined immunodeficiency (SCID) and its variants; Omenn syndrome;

[0061] Leukocyte adhesion deficiency: Wiskott-Aldrich syndrome; Chronic granulomatous disease; Chediak-Higashi syndrome; ZAP-70 deficiency; IL-7R, JAK3, RAG1 / RAG2 deficiency; Refractory autoimmune or autoinflammatory diseases: Severe multiple sclerosis; Refractory systemic lupus erythematosus; Progressive systemic sclerosis; Refractory Crohn's disease; Severe systemic juvenile idiopathic arthritis.

[0062] The invention may be better understood in light of the following examples.

[0063] Examples.

[0064] Example 1. Isolation of UC-MCS mitochondria and mitoception in CD34* cells

[0065] To isolate mitochondria for use in mitoception, the following protocol was performed: Mitochondrial isolation.

[0066] Cell preparation: We used umbilical cord mesenchymal stem cells (UC-MSCs) from passages 5-7. We stained the cells with mitochondrial dyes such as MitoTracker Green (MTG), Orange (MTO), or Deep Red FM (MTDR), following the manufacturer's instructions in each case. For mitochondria undergoing OXPHOS metabolism, no additional treatment of the UC-MSCs was necessary; for glycolytic metabolism, it was necessary to incubate the cells with oligomycin A (OLN A) for 24 hours before mitochondrial isolation.

[0067] Mitochondrial isolation was then performed using a commercial kit (Thermo Scientific, 89874) following the manufacturer's instructions. After final centrifugation, the pellets containing mitochondria (MT) were collected.

[0068] In each case, we confirmed the presence of mitochondria using transmission electron microscopy (TEM) to visualize mitochondrial morphology. Additionally, we performed Western blot analysis to detect specific mitochondrial proteins, such as TOM20. We also used flow cytometry to confirm mitochondrial-specific markers and dyes.

[0069] For functional assessment of mitochondrial cells (MTs), we measured ATP production using the CelITiter-Glo® luminescent cell viability assay. Simultaneously, we assessed mitochondrial membrane potential (A^Pm) using A^Pm-dependent dyes such as MitoTracker Orange (MTO). To perform mitoception, we used freshly isolated mitochondrial pellets from UC-MSCs, which showed a positive functional assessment. We incubated the mitochondrial pellets with human UCB-CD34 cells. +(HSPC) in different cellular proportions, this means that the isolated mitochondria of 2x10 6 UC MSCs are combined with, for example, 2xl0 6 CD34 cells + This corresponds to a 1:1 ratio. Incubation is carried out for 24 hours. Different ratios of mitochondria (relative to their cells of origin) with recipient cells were tested: 1:10, 1:5, 1:3, and 1:1. (mito: CD34 cells) +) .

[0070] We confirmed mitochondrial transfer using confocal laser scanning microscopy (CLSM) to visualize donor-derived mitochondria in recipient cells. Additionally, we performed flow cytometry to analyze mitochondrial uptake in different hematopoietic subpopulations of CD34+ cells. + (e.g., HSC and HPC).

[0071] This protocol guarantees the isolation of functional mitochondria and their transfer to target cells. The cells obtained are CD34+ cells. +HSPCs enriched in MT from UC-MSCs in OXPHOS metabolism have improved properties for grafting in patients with hematopoietic diseases.

[0072] All cultures performed in the examples were carried out at 37°C, atmosphere: 5% CO2, in humidified air; 5% O2, StemSpan™ culture medium, in the case of CD34 cells + The medium was further supplemented with cytokines (thrombopoietin (TPO), stem cell factor (SCF), flt3 ligand (FLT3), and interleukin-6 (IL-6)). The concentrations of the cytokines used in the final medium were as follows: SCF: 50 ng / mL; TPO: 10 ng / mL; FLT3: 50 ng / mL; and IL-6: 10 ng / mL.

[0073] Example 2. Contact-dependent mitochondrial transfer

[0074] We isolated CD34 cells + umbilical cord blood cells (UCB) were obtained using a commercial positive selection kit and cultured in Stem Span™ medium supplemented with cytokines.

[0075] CD34 cells + (HSPC) were cultured with different ratios of whole UC-MSC cells labeled with the mitochondria-specific fluorescent dye MitoTracker (1:1 or 3:1) or with mitochondrial isolates derived from those UC-MSC cells (1:10, 1:5, 1:3 and 1:1), as per Example 1. After 24 hours, MitoTracker levels were examined in the CD34 cell subpopulations + receptors were identified by confocal laser scanning microscopy (CLSM) and flow cytometry (Fig. 1). In co-culture, we observed that CD34 cells + They acquired Mitotracker+ fluorescence by CLSM after 24 hours of co-culture (Fig. IB). This result was confirmed by flow cytometry, showing that both stem cells and CD34 cells + Progenitor cells expressed donor-derived Mitotracker+ mitochondria when cocultured with UC-MSCs (Fig. 1C-F). The mother cell population (HSC) was determined to be CD34 + CD38 _and the progenitor cells (HPC) such as CD34 + CD38 +In both populations, mitochondrial transfer was dose-dependent, as the higher proportion of UC-MSCs showed a tendency to increase MitoT (Fig. 1C-F). Furthermore, HPCs, in both co-culture ratios, produced a higher percentage of Mitotracker+ (38.9% and 54.9%; Fig. 1E) compared to HSCs (32.5% and 42.8%; Fig. 1C), suggesting greater permissiveness of HPCs to MitoT. This same pattern was observed when analyzing the mean fluorescence intensity (MFI) of both subpopulations (Fig. 11 and 12F). The MFI of HSCs and HPCs increased significantly with a higher density of umbilical cord stem cells (UC-CMCs) (Mother: 1:1 - 247.3; 3:1 - 417.3; Progenitor: 1:1 - 745.6; 3:1 - 913.8) compared to the condition without co-culture. The MFI of Mitotracker+ was higher in progenitor cells after MitoT than in stem cells, suggesting that progenitor cells have a greater capacity to acquire donor-derived mitochondria.

[0076] Example 3. Mitoception: an artificial in vitro tool to study the effect of mitochondrial transfer in CD34* cells.

[0077] We worked with functional mitochondrial isolates, which we incubated for 24 hours with Mitotracker+ isolates derived from the donor with mitochondria in CD34 cells + recipients (Fig. 1A). We observed donor-derived Mitotracker+ mitochondria in CD34 cells + using CLSM, which confirmed mitochondrial donation (Fig. IB). By analyzing the profile of the CD34 surface marker graph + (green) and exogenous mitochondria derived from UC-MSCs (red), confirming mitochondrial internalization in target cells. The presence of the MitoTracker fluorescent signal in CD34 cells +It was detected just 5 hours after artificial administration of MitoT and increased 2.33-fold 24 hours after donation. Therefore, 24 hours was used as the time point for the remaining experiments.

[0078] Similar to co-culture, artificial mitochondrial transfer in hematopoietic stem cells (CD34 cells) was observed. + CD38", HSC) (Fig. 1C, D) and hematopoietic progenitor cells (CD34 cells) + CD38 + HPC) (Fig. IE, F) was dose-dependent and increased with higher mitochondrial content. Progenitor cells (CD34 cells) + CD38 + ) Mitotracker+ showed a higher percentage (1:1 - 35.5 and 49.8, respectively) and a higher IFM (1:1 - 433.3 and 183.0, respectively) than stem cells (CD34 cells) + CD38). These results were confirmed by CLSM of CD34 cells + CD38 _ or CD34 + CD38 +pre-sorted after 24 hours of MitoCeption in a 1:1 ratio (Fig. 1G). Quantification of HSCs and MitoTracker+ HPCs showed that a higher percentage of HPCs (57.8%) accepted exogenous mitochondria compared to HSCs (33.5%). Further analysis of hematopoietic progenitor subpopulations revealed that granulocyte megakaryocyte progenitor cells, GMP CD34 + CD38 + CD123 + CD45RA + These were the subpopulation that presented the highest percentage of MitoTracker+ (1:1 - 64.2%) and the greatest change in the fusion index (MFI) (1:1 - 4.8 times). On the other hand, the megakaryocyte and erythrocyte progenitor cells MEP CD34 + CD38 + CD123 CD45RA” showed the lowest percentage transfer yield (1:1 - 43.2%) and the lowest fusion index (MFI) change (1:1 - 42.9 times).

[0079] Example 4. Modulation of mitochondrial activity with oligomycin A.

[0080] Different concentrations (2.5, 5, and 10 pg / ml) and incubation times (4, 12, and 24 hours) of oligomycin A (OLN-A) were used to evaluate its effect on the induction of apoptosis in UC-MSCs and mitochondrial metabolism. Neither the concentrations used nor the incubation times led to a decrease in MSC viability. A 4-hour treatment with 5 pg / ml of OLN-A was sufficient to significantly reduce total ATP production in UC-MSCs (0.44-fold). We confirmed the decrease in oxidative phosphorylation (OXPHOS) metabolism using a mitochondrial stress test performed with Agilent Seahorse XF technology. UC-MSCs treated with OLN-A showed a significantly reduced baseline OCR and ATP-linked OCR compared to untreated MSCs.We assessed metabolism using quantitative, minimally invasive, dye-free fluorescence intensity-of-life microscopy (FLIM) to detect changes in the nicotinamide adenine dinucleotide (NAD+ / NADH) ratio. NAD+ / NADH are the main determinants of redox status in the cell, as NAD+ drives cytosolic ATP production via glycolysis and mitochondrial ATP production via oxidative phosphorylation (OXPHOS). FLIM detects the autofluorescence of these molecules and measures fluorescence decay rates (T) to assess changes in cellular metabolism in contrast to their oxidized, non-fluorescent forms. A cell favoring a glycolytic program exhibits a higher ratio of free NADH to protein-bound NADH compared to a cell preferentially undergoing oxidative phosphorylation (OXPHOS).Furthermore, the fluorescence lifetime of free NADH is significantly shorter (~0.4 ns) than that of protein-bound NADH, providing a quantitative assessment opportunity to evaluate a cell's metabolic state. Therefore, a metabolic spectrum can be defined as one in which glycolysis is at one end (high free:bound NADH ratio) while oxidative phosphorylation is at the other end (low free:bound NADH ratio). OLN-A-treated UC-MSCs showed a significantly shorter T0 after 1 hour (0.77) and 4 hours (0.25) of OLN-A treatment, suggesting that OLN-A-treated UC-MSCs are located at the glycolytic end of the metabolic spectrum.

[0081] Example 5. Mitochondrial donation replenishes the mitochondrial mass of CD34* cells and maintains low mitochondrial function.

[0082] We hypothesized that mitochondrial transfer can affect not only the metabolic parameters of hematopoietic subpopulations, but also their functional parameters, thereby enhancing the efficiency of UCB-HSC transplantation. First, we examined the effect of mitochondrial respiration and mitochondrial transfer derived from cells in a glycolytic metabolic state (MITO glyco) on the mitochondrial content and membrane potential of CD34 cells. + As mentioned earlier, CD34 cells + Cells capable of self-renewal and differentiation into multilineages prefer glycolysis to oxidative phosphorylation (OXPHOS) for energy production. This is because glycolysis allows for limited ROS production and promotes the potential of CD34+ cells +After inactivation of mitochondrial respiration by oligomycin A (OLN-A), the different isolates containing mitochondria were incubated for 24 hours with CD34 cells + (Fig. 4A).

[0083] Mitochondrial content was examined in the total CD34 population + using transmission electron microscopy. Cells that received mitochondria derived from untreated UC-MSCs (CD34 cells) + + MT oxphos) had an average of 21 mitochondria per cell, while those that received mitochondria from UC-MSCs treated with OLN-A (CD34 cells) + + MTglico) had a significantly lower mitochondrial content with an average of 5 mitochondria per cell, even lower compared to those that did not receive mitochondria (8 mitochondria; CD34 cells + (alone). Despite their higher mitochondrial content, CD34 cells ++ MT oxphos did not show a significant alteration in the mitochondrial area (0.15 pm 2 ) compared to HSPC alone (0.18 pm 2 Changes in mitochondrial parameters, such as size or mass, may indicate fission processes, mitochondrial stress, stress in ATP production, or imbalance in ionic homeostasis.

[0084] Next, we examined whether mitochondrial donation had any effect on A^Pm (mitochondrial membrane potential). Twenty-four hours after mitochondrial transfer, the CD34 populations + They were stained with the mitochondrial membrane potential dye TMRE. TMRE is a positively charged fluorescent dye that accumulates in mitochondria due to their negative charge. As expected, cells with long-term reconstitution activity, or LT-HSCs, defined by CD34 + CD38 CD133 +They maintain a low A^Pm, or depolarized mitochondria (Fig. 2E-J), since they do not sequester TMRE, resulting in a reduction of the MFI (mean fluorescence intensity) of TMRE. There was a significant (3-fold) increase in the percentage of low A^Pm LT-HSCs in the group that received MToxphos compared to the control group and the group that received MTglico (Fig. 2E). LT-HSCs at the low A^Pm threshold did not show a change in the MFI of TMRE. However, those that showed a high A^Pm threshold showed a significant increase upon receiving MTglico (Fig. 2F, G). Similarly, cells with short-term reconstitution activity or ST-HSCs showed a significant increase in the percentage of low A^Pm with MToxphos treatment, and at the same time an increase in the percentage and MFI of high A^Pm HSCs in the MTglico-treated group (Fig. 2H-J).Previous research has reported that LT-HSCs exhibit low mitochondrial activity concomitant with a low A^m. In fact, when HSCs with a low A^Pm were classified and transplanted into an irradiated murine model, these cells showed increased engraftment capacity, suggesting that they are enriched with LT-HSCs. These results suggest that mitochondrial donation from MToxphos promotes an increase in mitochondrial content without resulting in increased mitochondrial size or hyperpolarization of the mitochondrial membrane potential in CD34 cells. + CD38 CD133 + all of these are characteristics of the graft and the quiescence of CD34 cells + .

[0085] Example 6. OXPHOS-derived mitochondrial donation promotes CD34 cell expansion + (HPC).

[0086] To assess whether mitochondrial donation has effects on expansion while maintaining primitive CD34 phenotypes +HSPC and HCS, we cultured CD34+ cells (HSPC) obtained from fresh cord blood after mitochondrial transfer or MitoCeption as performed in example 1. We worked with cells transfected with MITOoxphos, MITOglico and untransfected control, distinguishing between two types of recipient cells: early reconstitution HPC (CD34 + CD38 + CD133 + ) (Fig. 3A) and those of long-term reconstitution HSC (CD34 + CD38 CD133 + (Fig. 3B). After a 7-day culture, we evaluated the expression of CD34, CD38, and CD133 in the different subpopulations by flow cytometry. Unlike the control group and the group that received MITOglico, the human papillary cell (HPC) culture that received MITOoxphos showed a 40-fold increase in CD34 cell expansion. +(HPC) on day seven, maintaining the phenotype (Fig. 3A); in contrast, the group that received MITOglico fared worse than the control group without mitochondrial donation. Furthermore, the expansion of long-term reconstitution cells (HCCs) (CD34 + CD38 _ CD133 + The expansion of cells treated with MITOoxphos showed no significant differences compared to the control without mitochondrial donation, but was substantially better than the expansion of cells treated with MITOglico (Fig. 3B). Notably, this increase in early progenitors was not accompanied by a decrease in stem cell populations (Fig. 3A-B).

[0087] In accordance with these results, we used single-cell sequencing clustering to analyze cell populations. The process involved isolating individual cells, sequencing their transcripts, and then performing computational clustering to identify distinct cell populations based on gene expression profiles. This approach allows for high-resolution analysis of cellular heterogeneity and lineage relationships. Our clustering analysis revealed that mitochondrial transfer markedly increases the proportion of cells within the hematopoietic stem cell (HSC) pool, as well as the multipotent progenitor (MPP) pool (Fig. 3C). These findings show that mitochondrial transfer with MITOoxphos promotes the expansion or preservation of these critical stem and progenitor cell populations.

[0088] Example 7. Mitochondrial transfer in differentiation

[0089] CD34 cells + They have the capacity to repopulate the entire hematopoietic system. In vitro assays have been developed to evaluate the capacity of CD34 cells +To differentiate and assess their function, tests such as the colony-forming unit (CFU) assay are used. The CFU evaluates the ability of a fixed number of cells to form different types of colonies in a semisolid medium supplemented with various cytokines after approximately 10–14 days. The number of colonies and their morphology reflect the cells' competence to differentiate and proliferate. Depending on the progenitor lineage, the colonies exhibit different characteristics, which can be identified as early progenitor colony-forming units (CFU-GEMM): granulocytes, erythrocytes, monocytes, megakaryocytes; intermediate progenitor colony-forming units (CFU-GM): granulocytes, monocytes; or late progenitor colony-forming units (CFU-E): erythroid burst-forming units (BFU-E), granulocytic colony-forming units (CFU-G), and monocytes (CFU-M).

[0090] We performed a CFU assay with 200 freshly isolated phenotypic HSPCs using a CD34 enrichment kit + After 10–12 days, the colonies were visualized under a microscope and counted manually. We found that mitochondrial treatment of HSPCs with MITOglico decreased the colony size of CFU-GEMM and CFU-GM. In addition, CFU-GEMM exhibited less reddish pigmentation. Mitochondria derived from UC-MSCs without MITO oxphos treatment had no effect on colony morphology or size. We then assessed total and type-specific colony counts (Fig. 3F-I). MITO oxphos increased the total number of colonies (71) compared to CD34 cells. +Solas (49) and MITOglico (39; Fig. 4A). Further analysis revealed that mitochondria derived from untreated UC-MSCs exhibited the highest percentage of CFU-GEMM (44.24%), but the lowest CFU-M (11.42%), suggesting a bias toward early progenitors (Fig. 3B-D). MITOglico showed a bias toward late progenitors of the myeloid lineage, as well as a reduced capacity to produce early progenitors (Fig. 3B-D). Therefore, MITOoxphos transfer promotes the expansion of both proliferating and non-proliferating HSCs, while also enhancing their ability to differentiate into early progenitors.

[0091] Example 8. Mitochondrial transfer in ROS relief

[0092] Reactive oxygen species (ROS) production has been widely associated with decreased hematopoietic stem cell (HSC) function, induced HSC exhaustion, and the promotion of HSC senescence. ROS are considered byproducts of mitochondrial respiration. Therefore, mitochondria are considered the primary site of ROS production. In this study, we assessed ROS relief in hematopoietic stem cell populations following stimulation with menadione, an oxidative stress inducer. We found that, following menadione treatment, mitochondrial-deficient HSCs and HPCs significantly increased the MFI of CM-H2-DCFDA, a total ROS stain (Fig. 5A, B). Interestingly, when HSCs received MITOoxphos (2.2-fold) and MITOglico (2.2-fold), ROS accumulation was significantly decreased in HSCs (Fig. 5A), but not in HPCs (Fig. 5B). Interestingly, a second population only emerged in hematopoietic stem cells (HSCs) that received MITOoxphos.This population showed a CM-H2-DCFDA MFI similar to that of cells not treated with menadione, suggesting greater ROS relief. A decrease in ROS production promotes the maintenance of HCS, as they can act as secondary molecules responsible for regulating various physiological functions, such as differentiation and self-renewal.

[0093] Conclusion

[0094] Mitochondrial transfer of untreated UC-MSC-derived mitochondria with MITOoxphos mimics the metabolism of quiescent HSCs by promoting glycolysis and reducing mitochondrial ATP production. Furthermore, HSCs receiving MITOoxphos exhibit greater expansion of both proliferating and non-proliferating HSCs, as well as enhanced differentiation capacity into early progenitors. Finally, MITOoxphos contributed to the reduction of ROS following oxidative stress induction, suggesting that mitochondrial transfer is a powerful tool for improving HSC fate and function in vitro.

[0095] Example 9. CD34* cell grafting in secondary hematopoietic organs: peripheral blood. To study the functionality of HSPCs after mitochondrial donation, we performed an in vivo repopulation assay with CD34 cells. +Complete mitochondrial cell lines were obtained from non-irradiated neonatal NOD-SCID (non-obese diabetic severe combined immunodeficiency) infants (1 to 3 days old) by intrahepatic injection (Fig. 6A). After 6 weeks, we performed a peripheral blood smear to examine the presence of human hematopoietic cells (CD45 + ) in the receptor mice. We observed that, as early as week 6, the CD34 cells + Cells treated with MITOoxphos (5.71%) showed a significantly greater short-term engraftment and repopulation capacity (Fig. 6 C, D). This pattern persisted until week 8, where CD34 cells + Cells that received MITOoxphos showed greater engraftment (20.5%) compared to those that did not receive mitochondria (6.96%) and those that received MITOglico (3.26%; Fig. 6 E, F). Similar to the in vitro functional assay, MITOglico reduced the functional capacity of HSPCs to produce daughter cells.

[0096] In addition, we studied the repopulation capacity of HSPCs to produce mature blood cells. MITOoxphos showed a tendency to increase the percentage of mature blood cells, such as CD3 (Fig. 6G), CD4 (Fig. 6H), and CD19 (Fig. 61) lymphocytes. As expected, MITOglico showed the opposite trend, significantly decreasing the percentage of all types of mature human blood cells. The presence of non-classical and intermediate CD8 monocytes was not detected under any condition. Classical monocytes showed a tendency to increase in HSPCs that were MITOoxphos-treated.

[0097] Example 10. Hematopoietic stem cell grafting into secondary hematopoietic organs: the spleen. Since the spleen has the capacity to host extramedullary hematopoiesis, we examined its hematopoietic content. Human hematopoietic cells (CD45) were found. +) in the spleen under all three conditions (Fig. 7A, B). However, HSPCs that received MITOoxphos showed greater graft adhesion (47.9%) than HSPCs alone (18.5%) and HSPCs with MITOglico (25.9%).

[0098] To determine whether other hematopoietic lineage populations showed differences between groups, we quantified the relative CD3 subpopulations + , CD4 + , CD8 + , CD19 + and monocytes. MITOoxphos was shown to significantly increase the total CD45 cell content + compared to HSPCs alone (~2.58 times) and MITOglico (~1.84 times) (Fig. 5A, B). Surprisingly, the spleen was the only organ where HSPCs receiving MITOglico showed better engraftment than the control group. Similar to peripheral blood, MITOoxphos promoted, although not significantly, an increase in the percentage of CD3 lymphocytes +humans. However, we were unable to observe other mature blood cells, such as CD4 + and CD8 + Although we observed the presence of CD19 + and classical monocytes. Mitochondrial transfer did not provide any advantage. Finally, we were unable to find any non-classical or intermediate monocytes in the spleen.

[0099] Example 11. Hematopoietic stem cell graft (CD34 + , HSPC) in the main hematopoietic niche: the bone marrow

[0100] The hematopoietic niche of the bone marrow is the primary site of hematopoietic stem cells (HSCs) and early hematopoietic progenitors. Therefore, we examined the engraftment and repopulation potential of the primary HSC microenvironment for mature hematopoietic populations (CD45+). + ), parents (CD45 + / Lin / CD34 + CD38) and stem cells (CD45 + / Lin7CD38 / CD34 + CD133 +). We observed that cells transfected with MITOoxphos significantly increased the presence of huCD45 + (Fig. 8A, B) compared to mice that received HSPC injections from the other groups. Therefore, we analyzed the lineage composition of the hematopoietic population. MITOoxphos mice showed a tendency to increase the content of more immature phenotypes, such as huLineage- (huLin-) and total huHPC, compared to other HSPC groups. However, no benefit of mitochondrial transfer was detected in the percentage of huHSCs after injection. Further analysis of the progenitor subpopulations revealed that only myeloid progenitors (CMPs) increased when HSPCs received MITOoxphos, but not the content of MEPs or GMPs.

[0101] Conclusion

[0102] These results confirm in vitro observations of improved HSPC function following mitochondrial transfer. OXPHOS-derived mitochondrial transfer promotes faster engraftment in NOD-SCID mice as early as week 6. Furthermore, an improvement in CD34 engraftment capacity was observed. + HSPC, reflected in the increase in CD45 cells + in medullary and extramedullary hematopoietic organs, suggesting a beneficial effect of mitochondrial transfer.

Claims

Claims 1. CD34 hematopoietic stem cell + modified CHARACTERIZED because the cell is enriched with mitochondria isolated from mesenchymal stem cells that are in oxidative phosphorylation metabolism.

2. CD34 cell + modified in accordance with clause 1 CHARACTERIZED in that it expands in cell culture without modifying its HSPC progenitor cell phenotype.

3. CD34 cell + modified in accordance with clause 1 CHARACTERIZED in that it has an improved grafting capacity for short and long-term reconstitution.

4. CD34 cell + modified in accordance with clause 1 CHARACTERIZED in that it has greater resistance to oxidative stress (ROS) compared to CD34 cells + are modify.

5. CD34 cell + modified in accordance with clause 1 CHARACTERIZED in that it has a higher proportion of bone marrow graft.

6. Method to improve CD34 cell properties + CHARACTERIZED in that it comprises: a) obtaining a mesenchymal cell culture in early passages, in the metabolic state of oxidative phosphorylation; a) optionally extracting the mitochondria from the MSC culture, obtaining a pellet rich in mitochondria of MSCs in oxidative phosphorylation (MITOoxphos); b) in parallel obtaining a CD34 cell culture + where these cells do not have deficiencies in their own mitochondria; c) incubate CD34 cells + either with the mesenchymal cells from step a) or with the mitochondria-rich pellet from step a1a); and d) obtain CD34 cells + enriched in mitochondria with improved properties of clause 1; where all stages are carried out under human cell culture conditions for use in cell therapy.

7. Method in accordance with clause 6 CHARACTERIZED in that the CD34 cells +Mitochondria-enriched cells have at least 50% more mitochondrial content compared to untreated cells.

8. Method according to clause 6 CHARACTERIZED in that in step c) the ratio between the CD34 cells + and the mitochondria ratio is between 1:1 to 1:5 mitochondrial donor cells, in relation to CD34 cells + .

9. Method according to clause 8 CHARACTERIZED in that in step c) the incubation of CD34 cells + and MITOoxphos develops for between 12 and 36 hours.

10. Method according to clause 9 CHARACTERIZED in that the incubation takes place for 24 hrs.

11. Pharmaceutical composition CHARACTERIZED in that it comprises a suspension of CD34+ cells enriched with mitochondria isolated from mesenchymal stem cells that are in oxidative phosphorylation metabolism, according to claim 1, in a pharmaceutically acceptable vehicle.

12. Pharmaceutical composition of clause 11 CHARACTERIZED because it is used in the treatment of hematopoietic and autoimmune diseases.

13. Pharmaceutical composition in accordance with clause 12 CHARACTERIZED in that it is used in bone marrow transplantation.

14. Pharmaceutical composition in accordance with clause 12 CHARACTERIZED in that it is used in bone marrow transplantation as part of the treatment of leukemia, myelomas, lymphomas, autoimmune diseases or other blood diseases.

Images